Epitopes: Types, B-cell vs T-cell Recognition, and Epitope Spreading

A single virus particle can trigger dozens of different antibodies at once, each one locking onto a slightly different spot on the same virus. Denature that virus with heat, and some of those antibodies suddenly stop working while others still bind perfectly. Why?

Because antibodies do not recognize a whole antigen. They recognize small, specific patches of it, and some patches survive denaturation while others are destroyed by it. Those patches are called epitopes, and understanding them explains why one antibody can be lost to heat while another is not, why B cells and T cells "see" completely different parts of the same microbe, and why the immune system sometimes turns on the body's own tissue.

Introduction

Epitopes, also known as antigenic determinants, are the immunologically active discrete sites on the antigen molecule that physically bind to antibodies, B-cell receptors, or T-cell receptors.

When an antibody binds to an antigen, it isn’t binding to the entire antigen but to a segment of that antigen known as an epitope. The part of an immunoglobulin that binds and fits the epitope is called a paratope​. Paratope is located at the tip of the variable region of an immunoglobulin, in its antigen-binding site. This paratope is only capable of binding with one unique epitope.

B cells can recognize an epitope alone but T cells can recognize an epitope only when it is associated with an MHC molecule on the surface of a self-cell (either an antigen-presenting cell or an altered self-cell).

An antigen can carry one or many epitopes. Most antigens carry many, meaning they are multivalent. B-cell epitopes, the ones recognized by antibodies, are generally small, on the order of 5 to 8 residues, though their size varies. T-cell epitopes are peptides of defined length set by the MHC molecule that presents them: roughly 8 to 10 amino acids for MHC class I, and roughly 13 to 17 amino acids for MHC class II. Keep these numbers in mind, because the size difference is tied directly to how each cell type recognizes antigen, which the next section explains.

There may be a presence of related antigens across various species. Related antigens have some epitopes in common but some that are different. Related antigens are also referred to as cross-reacting antigens because antibodies targeted to one antigen are able to react with all other antigens carrying the same epitope.

B-cell epitopes vs T-cell epitopes: the key distinction

The single most important idea about epitopes is that B cells and T cells recognize fundamentally different things, even on the same antigen. Getting this distinction clear resolves most confusion in this topic.

A B-cell epitope is recognized directly, in its native three-dimensional shape, by an antibody or B-cell receptor. Because the antibody meets the antigen in its folded form, B-cell epitopes are usually on the exposed, accessible surface of the molecule. They can be made of protein, carbohydrate, lipid, or nucleic acid. And critically, they depend on shape, so denaturing the antigen can destroy them.

A T-cell epitope is never recognized in its native form. The antigen must first be broken into peptide fragments inside a cell, and those fragments must be displayed on an MHC molecule for a T cell to recognize them. This has three consequences that follow logically:

First, T-cell epitopes are almost always peptides, because only peptides are processed and loaded onto MHC. Carbohydrate and lipid epitopes, which B cells handle easily, are largely invisible to conventional T cells.

Second, T-cell epitopes can come from buried, internal parts of a protein, precisely the regions a B cell could never reach on the intact molecule, because processing unfolds and chops the protein before presentation.

Third, T-cell epitopes depend on sequence, not on the folded shape, because the peptide is stretched out in the MHC groove. Denaturation does not destroy a T-cell epitope the way it destroys a conformational B-cell epitope.

This is why the two branches of the immune system are described as suited to different environments: B cells and antibodies patrol the outside surfaces of intact pathogens, while T cells inspect fragments of what is happening inside cells.

Functions of Epitopes

Epitope recognition by B-cell and T-cell is central to humoral and cell-mediated immune response.

The humoral branch (B cells) recognizes an enormous variety of epitopes (also referred to as B-cell epitopes): those displayed on the exposed regions of bacteria or viral particles, as well as those displayed on soluble proteins, glycoproteins, polysaccharides, or lipopolysaccharides that have been released from invading pathogens.

When B cells are exposed to T-dependent antigens, they get activated and undergo class switching, affinity maturation, and differentiate into plasma cells. Plasma cells produce large amounts of antibody specific for the epitope recognized by their immunoglobulin receptor.

The cell-mediated branch (T cells) recognizes protein epitopes (also referred to as T-cell epitopes) displayed together with MHC molecules on self-cells, including altered self-cells such as virus-infected self-cells and cancerous cells. As T cells recognize only the processed peptides, those epitopes may be located on those regions (e.g., internal proteins) which are inaccessible to B-cells. Thus, each branch of the immune system uniquely suited to recognize antigen in a different environment.

T-cell epitopes are presented by class I (MHC I) and II (MHC II) MHC molecules that are recognized by two distinct subsets of T-cells, CD8, and CD4 T-cells, respectively. Subsequently, there are CD8 and CD4 T-cell epitopes. T-cells become cytotoxic T lymphocytes (CTL) following T CD8 epitope recognition. Meanwhile, primed CD4 T-cells become helper (Th) or regulatory (Treg) T-cells.

Figure: B-cell (left) and T-cell (right) epitope recognition (Image source-Ref-1)

Types of Epitopes

The antigenic determinants (epitopes) are divided into two categories based on their structures and interaction with the paratope.

• Linear epitopes
• Conformational epitopes

Figure: Linear and conformational B-cell epitopes. Linear B-cell epitopes (a) are composed of sequential/continuous residues, while conformational B-cell epitopes (b) contain scattered/discontinuous residues along the sequence. (Image source-Ref-1)

This distinction has a practical consequence. Most B-cell epitopes on natural proteins are conformational, so they exist only while the protein is folded. This is why some diagnostic tests must use native, non-denatured antigen to detect the right antibodies, and why heat or chemical denaturation can cause a true antibody to appear falsely negative. Linear epitopes, by contrast, survive denaturation and are the ones typically used in peptide-based assays and vaccines.

Epitope Spreading

Epitope spreading explains one of the puzzles of autoimmune disease: why an immune response that starts against a single target can, over time, broaden to attack many. It begins as a response to one epitope and spreads to others, either on the same molecule or on neighboring ones.

Epitope spreading or ‘determinant spreading’ denotes ‘development of immune responses to endogenous epitopes secondary to the release of self-antigens during a viral infection or a chronic autoimmune or inflammatory response’. In such conditions, sequestered autoantigens are exposed to autoreactive T cells causing autoimmune disease.

In an animal model study, it was found that animal infected with encephalomyelitis virus shows multiple sclerosis-like disease because self-reactive T cells react with cellular antigens rather than the antigens of the virus.

Mechanism of Epitope spreading

Figure: Mechanism of epitope spreading (Image source: Ref-4)

A polypeptide antigen (having multiple epitopes) is processed intracellularly by an antigen-presenting cell and a small peptide fragment is presented to a Th 1 cell. The T cell responds to the peptide by releasing cytokines which stimulates a B cell to produce antibodies specific to the peptide fragment and also express antigen-specific immunoglobulins on the cell surface.

Surface immunoglobulin of B cells subsequently recognize the intact antigen, internalize it, and process it. But, in this case, B cell presents a new peptide epitope via MHC class II to a T cell, thereby initiating the production of different antibodies to a new epitope of the same antigen.

This broadening of the immune response can target epitopes either within the same antigen (intramolecular spreading) or another antigen (intermolecular spreading). Multiple factors are involved in the induction of epitope spreading, such as:

• enhanced display of previously hidden antigenic determinants under the local inflammatory/cytokine environment
• Release of self-antigens following tissue damage
• Role played by B cells as antigen-presenting cells.

The clinical importance is this. In diseases such as multiple sclerosis, systemic lupus erythematosus, and pemphigus, the immune response measured early in disease targets fewer epitopes than the response measured later. As tissue damage releases more self-antigens, the response spreads to new epitopes, and the disease broadens and becomes harder to control. Epitope spreading is therefore not just a laboratory curiosity; it is part of why some autoimmune diseases progress.

Identification of Epitopes

With the availability of newer information about the specific roles played by epitopes and their potential use, research is ongoing to identify epitopes of complex antigens. Epitope identification is a costly and time-consuming process. It requires experimental screening of large arrays of potential epitope candidates.

Epitope identification can provide different benefits such as

• understanding disease etiology,
• immune monitoring,
• developing diagnosis assays,
• designing epitope-based vaccines

We hope to see breakthrough this is field of research.

How to remember

B for Big-picture, T for Torn-apart. B cells recognize the antigen whole, in its native shape (big picture). T cells only recognize it after it has been torn into peptides and loaded onto MHC.

Conformational needs the fold; linear survives the unfold. A conformational epitope exists only while the protein is folded, so denaturation destroys it. A linear epitope is a stretch of sequence, so it survives denaturation. If heat abolishes binding, the epitope was conformational.

Size ladder, small to large: B-cell epitope (~5–8) < MHC I peptide (~8–10) < MHC II peptide (~13–17). The T-cell numbers are set by the MHC groove, not by the T cell.

Spreading spreads outward: the response starts at one epitope and moves to more, like a crack spreading across glass. Intramolecular (same antigen) or intermolecular (a neighboring antigen).

Key exam facts in one table

Where students get confused

"An epitope and a paratope are the same thing." No, they are a matched pair. The epitope is on the antigen; the paratope is on the antibody. The paratope sits at the tip of the antibody's variable region and binds one specific epitope.

"T cells recognize whole antigens like B cells do." No. This is the core error. B cells recognize native, intact antigen directly. T cells only recognize a processed peptide displayed on an MHC molecule. A T cell will never bind a free-floating intact protein.

"T cells can recognize sugar and lipid epitopes." Generally no. Because T-cell recognition requires processing and MHC loading of peptides, conventional T cells respond to protein-derived peptides. Carbohydrate and lipid epitopes are mainly B-cell territory.

"Denaturation destroys all epitopes." No, and this is a useful discriminator. Denaturation destroys conformational B-cell epitopes but spares linear ones, and it does not abolish T-cell recognition, since the antigen was going to be unfolded and chopped up anyway.

"A bigger antigen just means a bigger epitope." No. A bigger antigen means more epitopes, not larger ones. Epitope size is fairly fixed; multivalency is about number, not size.

References and further readings

1. Sanchez-Trincado JL, Gomez-Perosanz M, Reche PA. Fundamentals and methods for T- and B-cell epitope prediction. J Immunol Res. 2017;2017:2680160. https://doi.org/10.1155/2017/2680160
2. Powell AM, Black MM. Epitope spreading: protection from pathogens, but propagation of autoimmunity? Clin Exp Dermatol. 2001;26(5):427–433. https://doi.org/10.1046/j.1365-2230.2001.00852.x
3. Cruse JM, Lewis RE, Wang H, eds. Antigens, immunogens, vaccines, and immunization. In: Immunology Guidebook. Academic Press; 2004:17–45.
4. Punt J, Stranford SA, Jones PP, Owen JA. Kuby Immunology. 8th ed. W.H. Freeman; 2019.